1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for tailing and amplifying non-polyadenylated RNA molecules, including microRNA, siRNA, tRNA, rRNA, synthetic RNA, and non-polyadenylated mRNA, such as mRNA from bacteria or fragments of prokaryotic or eukaryotic mRNAs such as may be encountered in degraded RNA samples.
2. Description of Related Art
The commonly used method for mRNA purification from eukaryotic cells, oligo-dT capture, is ineffective for capturing RNA molecules lacking poly(A) tails. Examples of RNA molecules that are not polyadenylated include microRNA, siRNA, tRNA, rRNA, synthetic RNA, and non-polyadenylated mRNA from prokaryotes. In addition, mRNA fragments, such as may be encountered in degraded RNA samples, may also lack poly(A) tails. The study of these important RNA molecules is hindered by inadequate methods for their isolation and amplification.
For example, small RNAs, such as microRNA (miRNA) and small interfering RNA (siRNA), have emerged as powerful post-transcriptional regulators of gene expression in many different organisms, thus making the analysis of small RNA molecules increasingly important. To generate scientifically accurate data, the analysis of small RNA molecules requires their quantitative purification and amplification. There is a need for novel methods and compositions that are capable of quantitatively purifying and amplifying these important molecules.
Another example of the difficulties encountered when analyzing non-polydenylated RNA molecules is the study of bacterial gene expression. The ability to evaluate global gene expression responses of bacterial pathogens during host cell infections is seen as crucial for identifying novel bacterial targets for therapeutic intervention and for a comprehensive understanding of bacterial pathogenesis. However, methods for in vivo whole genome expression analyses have not been fully realized. Genome-wide analysis of bacterial gene expression, following growth in the presence of host cells in vitro or in vivo, has been referred to as the “Holy Grail” of pathogen expression analysis (Sassetti and Rubin, 2002). Others have more recently reiterated that this remains the “ultimate goal” of pathogen gene expression analysis (Schoolnik, 2002; Conway and Schoolnik, 2003). The technical difficulties that hinder these studies include (1) purifying bacterial RNA free from large amounts of contaminating host cell RNA, and (2) purifying adequate amounts of bacterial RNA for DNA microarray analysis.
Bacteria respond rapidly to changes in their microenvironments. This quick adaptability enables them to survive and grow in all kinds of environmental conditions, including of course, eukaryotic host organisms. Many studies with bacterial pathogens have shown that bacteria exhibit altered gene/protein expression during interactions with host cells. In the past few years, host-induced changes in bacterial gene expression have been identified for Neisseria meningitidis (Taha et al., 1998; Grifantini et al., 2002; Dietrich et al., 2003), Mycobacterium tuberculosis (Triccas et al., 1999), Salmonella spp. (Eriksson et al., 2003; Valdiva and Falkow, 1997), Brucella suis (Boschiroli et al., 2002), and Legionella pneumophila (Abu Kwaik, 1998), to name a few. Analyses of bacterial gene expression during interactions with host cells will provide information on (1) the specific conditions of the host microenvironment (e.g., within phagocytic vacuoles, (Staudinger et al., 2002), (2) mechanisms and pathways used by bacteria in response to those microenvironments, (3) proteins that are essential for survival and growth in vivo, (4) novel vaccine candidates, and (5) novel targets for antibiotics.
Identifying bacterial genes whose expression is altered by host cells has not been easy, and microbiologists have used numerous techniques to address the challenge. These include mRNA-subtracted cDNA libraries (Scott-Craig et al., 1991; Plum and Clark-Curtiss, 1994), in vivo expression technology (IVET, (Mahan et al., 1993), signature tagged mutagenesis (Hensel et al., 1995), gene fusions with lacZ (Taha et al., 1998) and gfp (Triccas et al., 1999; Valdiva and Falkow, 1997; Boschiroli et al., 2002), differential display PCR (Abu Kwaik and Pederson, 1996), one- and two-dimensional electrophoresis of cell proteins (Abu Kwaik, 1998; Abshire and Neidhardt, 1993; Monahan et al., 2001), and cDNA selection (SCOTS-selective capture of transcribed sequences, (Graham and Clark-Curtiss, 1999). Many of these techniques have proven invaluable for the initial identification of specific bacterial genes whose expression is altered by interaction with host cells. However, all of these methods have limitations. Most require significant genetic manipulation (construction of mutants, libraries, gene fusions) and are labor intensive. Chief amongst the limitations is that none allows for quantification of genome-wide expression. Two-dimensional protein electrophoresis comes closest to allowing global expression analyses. However, at this point, it too is limited by resolution and sensitivity.
A handful of research studies have appeared in which investigators have overcome the technical difficulties described above. Investigators at Chiron and The Institute For Genomic Research (TIGR) used microarrays to analyze gene expression of Neisseria meningitidis following adherence to cultured epithelial cells (Grifantini et al., 2002). To reduce contaminating host-cell RNA, the epithelial cells were selectively lysed with saponin (a cholesterol-binding detergent), bacterial cells were harvested, and RNA was purified. Another group used SDS to selectively lyse macrophages containing phagocytized Salmonella enterica (Eriksson et al., 2003). RNA was then purified from the harvested bacteria. Staudinger et al. (2002) harvested total RNA from neutrophils with internalized E. coli. The authors reported that bacterial RNA was estimated to be ⅕ or more of the RNA isolated from the sample. Staudinger et al. used gene-specific primers for cDNA synthesis prior to E. coli array analysis.
Selective eukaryotic cell lysis followed by harvesting of bacteria is a useful method for increasing the relative amount of bacterial RNA isolated from microbe-host cell mixtures. It is imperative that RNA be stabilized prior to such treatments. If it is not, detergent treatment will likely alter the gene expression profile of the bacteria. In addition, selective detergent lysis of host cells is less effective with many Gram-negative bacteria that are easily lysed with such detergents (e.g., Yersinia enterocolitica). Furthermore, variable susceptibility of eukaryotic cells to saponin-mediated lysis makes optimization difficult. Selective eukaryotic cell lysis may be least useful with infected tissue samples that require vigorous homogenization to effect cell lysis. Nonetheless, selective lysis of host cells with detergents is a good idea that can be helpful for reducing excessive amounts of host cell RNA.
The use of gene-specific primers with bacterial arrays is another method (Staudinger et al., above) that has been used in hope of specifically priming bacterial RNA's. But, this results in the loss of ˜30% of hybridization signals on E. coli arrays (Khil and Camerini-Otero, 2002; Arfin et al., 2000).
A small percentage of bacterial mRNAs are poly(A)-tailed, but these are targeted for degradation and tend to be unstable. As a result, the commonly used method for mRNA purification with eukaryotic cells, oligo-dT capture, is ineffective for capturing bacterial mRNAs. Methods to polyadenylate bacterial mRNAs, thereby allowing for their purification by oligo dT-capture have been developed. Amara and Vijaya (1997) demonstrated that mRNAs in purified polysomes can be specifically polyadenylated and purified by oligo-dT capture. However, using their method Amara and Vijaya were not able to efficiently poly(A) tail the transcripts. For example, they reported that only 50% of the NS3 messages were polyadenylated. Wendisch et al. (2001) showed that the same process can be carried out with crude cell extracts.
As suggested above, several shortcomings are associated with the polyadenylation approach described in the prior art. Different mRNAs may be polyadenylated to different extents or not at all depending on the structure of their 5′ and 3′ ends (Feng et al., 2000). Polyadenylation in a cell lysate, followed by purification of RNA, will require inactivation of cellular RNAses so that transcripts are not degraded during the polyadenylation reaction. Optimizing the reaction to work reproducibly in many different bacterial cell lysates would likely be very difficult.
If methods of polyadenylating bacterial mRNAs are to be used in genome-wide expression analysis, it is important that as much as possible, if not all, mRNAs in a population of bacterial mRNA are poly(A) tailed, thereby ensuring that they will be representatively purified by oligo-dT capture and/or templates for the 1st strand cDNA synthesis reaction. However, using the standard reaction conditions for commercially available PAP enzymes and synthetic RNA transcripts, the inventors found that only about 90-95% of a transcript could be tailed.
Furthermore, it was also observed that with small mass amounts of RNA, such as those likely to be used for amplification (<100 ng), the tail lengths were extremely long—up to 9 kb in length. The inventors surmise that excessive tail-length could inhibit amplification reactions through several possible mechanisms: (1) reverse transcriptase may dissociate from the template during polymerization through extremely long homopolymeric A tracts; (2) dTTP may be effectively exhausted during 1st strand synthesis, slowing the reaction rate; (3) excess poly(A) may itself be detrimental to reaction kinetics; (4) UTP may become limiting during the in vitro transcription step; and/or (5) T7 polymerase may be hindered while incorporating long U tracts at the 5′ ends of antisense RNAs (aRNAs).